1. Field of the Invention
This invention concerns peptides, polypeptides, glycoproteins, their functional mutants, variants, analogs, and fragments useful for treatment and detection/diagnosis of C. parvum infections. In particular, this invention concerns competitive inhibition of the function of a Cryptosporidium protein/glycoprotein comprised of a protein with or without carbohydrates attached thereto. Additionally, this invention concerns DNA and RNA encoding the Cryptosporidium protein/glycoprotein, mutants, variants and analogs and fragments thereof, and methods for production of recombinant or fusion proteins for use in treatment and detection/diagnosis.
2. Background and Related Disclosures
The genus Cryptosporidium consists of Apicomplexan parasites that invade and develop within epithelial cells of the gastrointestinal, hepatobiliary and respiratory tracts of a wide variety of vertebrates including reptiles, birds and mammals. Cryptosporidium was recognized as a cause of animal disease for several decades before the first cases of human cryptosporidiosis were reported in 1976. However, it was not until 1982 that the magnitude of disease caused by this parasite in both AIDS patients and immunocompetent hosts began to be appreciated. Subsequently, Cryptosporidium has been found to be one of the most common causes of human diarrhea worldwide, and to be an increasingly recognized cause of diarrhea in children, animal care workers, and travelers. (Cryptosporidium and Cryptosporidiosis, Ed. Fayer, R., CRC Press, Boca Raton (1997)).
Large waterborne outbreaks of cryptosporidiosis caused by contaminated municipal water supplies in the U.S. or in the UK have been noted in the last ten years (N. Engl. J. Med., 320:1372 (1989), and 33:161 (1994)). The most recent outbreak in Milwaukee in April 1993 involved 400,000 persons and led to the subsequent deaths of more than 100 immunocompromised persons. Like a number of other waterborne outbreaks, the Milwaukee outbreak appears to have been due to contamination from farm or abattoir run-off and specifically to cryptosporidiosis among cows/calves. Additionally, nosocomial transmission in hospitals from patients to staff, patient to patient, and contaminated ice to patients and staff have also been well documented (J. Infect. Dis., 158:647 (1985)).
Investigation of waterborne and nosocomial outbreaks uncovered a number of biological characteristics of oocysts. First, the infectious dose of a parasite is very low. The ID.sub.50 for human volunteers with normal immune systems is 132 oocysts (N. Engl. J. Med., 332:855 (1995)). Second, infected hosts, for example calves, excrete large numbers of oocysts, on the order of 10.sup.10 /day. Third, the oocysts are fully sporulated and ready to infect when excreted. Fourth, the oocysts are environmentally hardy. They remain infectious in cool, moist areas for 3-4 months. They are not killed by chlorine levels achievable in drinking water. Fifth, the oocysts are quite small, 4-6 .mu.m, and are thus difficult to filter.
The clinical importance of cryptosporidiosis has increased markedly with the recognition of a life-threatening form of the disease in patients with immunodeficiency disorders such as AIDS, hypogammaglobulinaemia, and chemotherapeutic immunosuppression. The prevalence of cryptosporidiosis in AIDS patients in the U.S. is estimated to be 5-10% and in central Africa around 40%. Immunodeficient patients may have fulminant cryptosporidial diarrhea that may persist until death, whereas the diarrhea of immunocompetent patients is self-limited and rarely lasts more than 2-4 weeks. Cholera-like diarrhea is common in immunocompromised patients with reported losses of up to 17 liters per day. Hepatobiliary disease may result in severe abdominal pain and nausea. Removal of immunosuppression in chemotherapy patients leads to resolution of the diarrhea. Some AIDS patients with cryptosporidiosis may be able to eliminate the parasite in the presence of anti-retroviral therapy (Am. Intern. Med., 116:840 (1992)).
Among patients who develop disease, a quarter have CD4 counts greater than 209, suggesting that the disease may occur relatively early in the course of HIV disease (Am. J. Epidemiol., 144:807 (1996). Unfortunately, few details about the biology and molecular mediators of the disease process have been described and so far no effective therapy has been discovered.
Thus it would be very beneficial to have available a method for treatment of cryptosporidiosis.
U.S. Pat. No. 5,643,772 describes Cryptosporidium hybrid vector and transformed host cells specifically binding anti-Cryptosporidium antibodies. U.S. patent application Ser. No. 08/700,651 describes vaccines, antibodies, proteins, DNAs and RNAs for prophylaxis and treatment of Cryptosporidium parvum infections. U.S. patent application Ser. No. 08/827,171 describes vaccines, antibodies and proteins for prophylaxis and treatment of Cryptosporidium parvum infections. The patent and both applications are hereby incorporated by reference.
During the development of the disease, infective forms of Cryptosporidium, called sporozoites and merozoites, appear to adhere to the host cell and release the contents of anterior organelles (rhoptries, micronemes or dense granules) during the invasion process (Parasitol. Today, 8:28(1992)). Proteins involved in these events have in many instances been found to be the target of invasion blocking immunity in vitro and neutralization in vivo (Infect. Immun., 56:2538(1988)).
There is, therefore, a continuing need to have available methods for treatment and diagnosis of cryptosporidiosis as well as for detection of cryptosporidiosis in the environment. These methods also include techniques for reproducible propagation of Cryptosporidium protein/glycoprotein and for expression of Cryptosporidium protein/glycoprotein in large amounts, which antigen would provide a basis for production of competitive inhibitor molecules. In addition, these techniques would provide a source for DNAs and RNAs for production of protein/glycoprotein. There is also a need to have available methods for reproducible expression of a recombinant, engineered or otherwise modified protein for competitive inhibition of infection. In addition there is a need to utilize synthetic molecules serving the same function as the protein competitive inhibitor. This approach requires that a specific Cryptosporidium protein/glycoprotein is cloned and identified as a potential candidate through its ability to competitively inhibit infection.
It is therefore a primary objective of this invention to provide a method for treatment of cryptosporidiosis involving recombinant, engineered or otherwise modified protein, or substitutes which have the same function as Cryptosporidium GP900 protein/glycoprotein in competitively inhibiting infection. Additionally such proteins or substitutes would be used for detection/diagnosis of cryptosporidiosis through competitive inhibition.
All patents, patent applications and publication cited herein are hereby incorporated by reference.